A five-membered heteroaryl antifungal compound and use thereof

Abstract

The application relates to a kind of five-membered heteroaryl antifungal compounds and its application, the structural formula of the compound is as shown below:;Wherein, Ar is selected from the group consisting of,, and,;R1 It is selected from F and H;R2 It is selected from F and H;R3 It is selected from NO2, CN and H;R4 It is selected from CN and H;R5 It is selected from CN, F and H;L1 It is selected from-CH2O-, -OCH2- and-O-;T1 It is selected from N and CH.The application develops a series of five-membered heteroaryl compounds on the basis of APX001A structure, has good Gwt1 protein binding, can inhibit Gwt1 protein activity, thereby prevent GPI-AP synthesis, lead to the surface of fungi mannose protein cannot be crosslinked on cell wall, further destroy its adhesion host surface ability and cell wall integrity, play antifungal effect, have good inhibitory activity to candida, cryptococcus and aspergillus.

Description

Technical Field

[0001] This invention relates to the field of five-membered heteroaryl compounds, and more specifically, to a five-membered heteroaryl antifungal compound and its application. Background Technology

[0002] Invasive fungal infections (IFDs) are among the deadliest fungal infections, primarily caused by Candida, Aspergillus, and Cryptococcus, and can lead to infections in multiple tissues and organs, including the lungs and brain. Fungal cell walls are mainly composed of dextran, chitin, and mannose proteins. Glycosylphosphatidylinositol ovalbumin (GPI-AP) is anchored to the cell membrane and cell wall, mediating cross-linking between mannose proteins and dextran, and plays a crucial role in fungal cell wall synthesis, adhesion, and morphological transformation. Studies of fungal physiological structures have revealed that Gwt1 (a protein playing a vital role in fungal cell wall integrity) is a key acetyltransferase in the conserved glycosylphosphatidylinositol (GPI) post-translational modification pathway of eukaryotic cell surface proteins.

[0003] APX001A is an isoxazole-based small-molecule antifungal compound discovered by Eisai Co., Ltd. of Japan. Its structure is as follows:

[0004] This compound is a novel broad-spectrum antifungal agent that inhibits Gwt1. Inhibition of Gwt1 activity leads to the blockage of GPI-AP synthesis, preventing the cross-linking of fungal surface mannose proteins to the cell wall, thereby disrupting their ability to adhere to the host surface and cell wall integrity, thus exerting an antifungal effect. Therefore, this type of isoxazole small-molecule antifungal compound is a promising new antifungal compound. Its phase II clinical dosing design is as follows: intravenous injection, 1000 mg twice on the first day; 600 mg / day on the second and third days, followed by intravenous injection of 600 mg / day until day 42, or oral administration of 800 mg / day until day 42. The relatively high dosage is due to the poor solubility of APX001A, requiring it to be formulated as a prodrug for administration. The larger dosage after prodrug formulation also indicates the poor in vivo efficacy of APX001A.

[0005] In view of the above, this application is hereby submitted. Summary of the Invention

[0006] To address the aforementioned problems, this invention provides a five-membered heteroaryl antifungal compound and its applications. Based on the APX001A structure, a series of five-membered heteroaryl compounds have been developed, exhibiting good Gwt1 protein binding activity. These compounds inhibit Gwt1 protein activity, thereby preventing GPI-AP synthesis and preventing the cross-linking of fungal surface mannose proteins onto the cell wall. This disrupts the fungal's ability to adhere to the host surface and maintain cell wall integrity, thus exerting an antifungal effect. The compounds show good inhibitory activity against Candida, Cryptococcus, and Aspergillus, with a long half-life, low effective inhibitory concentration, and minimal side effects.

[0007] This invention is achieved through the following technical solution: In a first aspect, the present invention provides a five-membered heteroaryl antifungal compound, wherein the compound is a compound with the structural formula shown in formula (1) or a pharmaceutically acceptable salt, prodrug, or isotope derivative thereof: (1); Among them, R1 is selected from F and H; R2 is selected from F and H; R3 is selected from NO2, CN, and H; R4 is selected from CN and H; R5 is selected from CN, F, and H; L1 is selected from -CH2O-, -OCH2- and -O-; T1 is selected from N and CH.

[0008] In one specific embodiment, the structural formula of the five-membered heteroaryl antifungal compound is shown in A1 below: ; Where Ar is selected from R1 is selected from H; R2 is selected from H; R3 is selected from H; R4 is selected from CN; R5 is selected from H; L1 is selected from -CH2O-; T1 is selected from N.

[0009] The synthetic route for compound A1 is as follows: .

[0010] In one specific embodiment, the structural formula of the five-membered heteroaryl antifungal compound is shown in A2 below: ; Where Ar is selected from R1 is selected from H; R2 is selected from H; R3 is selected from H; R4 is selected from H; R5 is selected from CN; L1 is selected from -CH2O-; T1 is selected from N.

[0011] The synthetic route for compound A2 is as follows:

[0012] In one specific embodiment, the structural formula of the five-membered heteroaryl antifungal compound is shown in A3 below: ; Where Ar is selected from R1 is selected from H; R2 is selected from H; R3 is selected from NO2; R4 is selected from H; R5 is selected from H; L1 is selected from -CH2O-; T1 is selected from N.

[0013] The synthetic route for compound A3 is as follows:

[0014] In one specific embodiment, the structural formula of the five-membered heteroaryl antifungal compound is shown in A4 below: ; Where Ar is selected from R1 is selected from H; R2 is selected from F; R3 is selected from H; R4 is selected from H; R5 is selected from H; L1 is selected from -OCH2-; T1 is selected from CH.

[0015] The synthetic route for compound A4 is as follows: .

[0016] In one specific embodiment, the structural formula of the five-membered heteroaryl antifungal compound is shown in A5 below: ; Where Ar is selected from R1 is selected from F; R2 is selected from H; R3 is selected from H; R4 is selected from H; R5 is selected from H; L1 is selected from -OCH2-; T1 is selected from CH.

[0017] The synthetic route for compound A5 is as follows: .

[0018] In one specific embodiment, the structural formula of the five-membered heteroaryl compound is shown in A6 below: ; Where Ar is selected from R1 is selected from F; R2 is selected from H; R3 is selected from H; R4 is selected from H; R5 is selected from F; L1 is selected from -O-; T1 is selected from N.

[0019] The synthetic route for compound A6 is as follows: .

[0020] In one specific embodiment, the structural formula of the five-membered heteroaryl antifungal compound is shown in A7 below: ; Where Ar is selected from R1 is selected from H; R2 is selected from F; R3 is selected from H; R4 is selected from H; R5 is selected from F; L1 is selected from -O-; T1 is selected from N.

[0021] The synthetic route for compound A7 is as follows:

[0022] In one specific embodiment, the structural formula of the five-membered heteroaryl antifungal compound is shown in A8 below: ; Where Ar is selected from R1 is selected from H; R2 is selected from H; R3 is selected from H; R4 is selected from H; R5 is selected from H; L1 is selected from -CH2O-; T1 is selected from N.

[0023] The synthetic route for compound A8 is as follows: .

[0024] In one specific embodiment, the structural formula of the five-membered heteroaryl antifungal compound is shown in A11 below: ; Where Ar is selected from R1 is selected from H; R2 is selected from H; R3 is selected from CN; R4 is selected from H; R5 is selected from H; L1 is selected from -CH2O-; T1 is selected from N.

[0025] The synthetic route for compound A11 is as follows: .

[0026] Secondly, the present invention provides an application of a five-membered heteroaryl antifungal compound, wherein the five-membered heteroaryl compound is used to inhibit the activity of fungal Gwt1 protein.

[0027] In one specific embodiment, the fungi include Candida, Aspergillus, and Cryptococcus.

[0028] Thirdly, the present invention provides an application of a five-membered heteroaryl antifungal compound for the preparation of a drug for treating deep fungal infections.

[0029] Compared with the prior art, the present invention has the following advantages and beneficial effects: 1. The present invention provides a five-membered heteroaryl antifungal compound and its application. A series of five-membered heteroaryl compounds were developed based on the APX001A structure. These compounds have good Gwt1 protein binding activity and can inhibit Gwt1 protein activity, thereby preventing GPI-AP synthesis. This prevents the mannose protein on the fungal surface from cross-linking onto the cell wall, thereby destroying its ability to adhere to the host surface and the integrity of the cell wall, and thus exerting an antifungal effect. 2. The five-membered heteroaryl antifungal compound and its application provided in the embodiments of the present invention have good inhibitory activity against Candida, Cryptococcus and Aspergillus, good PK properties and in vivo antibacterial effect, and have low effective antibacterial concentration and few side effects. Detailed Implementation

[0030] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the embodiments. The illustrative embodiments and descriptions of this invention are only used to explain this invention and are not intended to limit this invention.

[0031] In the following description, numerous specific details are set forth in order to provide a thorough understanding of the invention. However, it will be apparent to those skilled in the art that these specific details are not necessary to practice the invention. In other embodiments, well-known materials or methods have not been specifically described in order to avoid obscuring the invention.

[0032] Throughout this specification, references to “an embodiment,” “an example,” or “an example” mean that a particular feature, structure, or characteristic described in connection with that embodiment or example is included in at least one embodiment of the invention. Therefore, the phrases “an embodiment,” “an example,” “an example,” or “an example” appearing in various places throughout the specification do not necessarily refer to the same embodiment or example. Furthermore, specific features, structures, or characteristics can be combined in one or more embodiments or examples in any suitable combination and / or sub-combination. The term “and / or” as used herein includes any and all combinations of one or more of the associated listed items.

[0033] The "range" disclosed in this application is defined by a lower limit and an upper limit. A given range is defined by selecting a lower limit and an upper limit, which define the boundaries of a particular range. Ranges defined in this way can include or exclude endpoints and can be arbitrarily combined; that is, any lower limit can be combined with any upper limit to form a range. For example, if ranges of 60–120 and 80–110 are listed for a specific parameter, it is understood that ranges of 60–110 and 80–120 are also expected. Furthermore, if minimum range values ​​of 1 and 2 are listed, and if maximum range values ​​of 3, 4, and 5 are listed, then the following ranges are all expected: 1–3, 1–4, 1–5, 2–3, 2–4, and 2–5. In this application, unless otherwise stated, the numerical range "a–b" represents a shortened representation of any combination of real numbers between a and b, where a and b are real numbers. For example, the numerical range "0~5" indicates that all real numbers between "0~5" have been listed in this article; "0~5" is simply a shortened representation of these numerical combinations. Furthermore, when a parameter is stated as an integer ≥2, it is equivalent to disclosing that the parameter is, for example, an integer such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc.

[0034] Unless otherwise specified, all steps in this application may be performed sequentially or randomly, preferably sequentially. For example, the method includes steps (a) and (b), indicating that the method may include steps (a) and (b) performed sequentially, or it may include steps (b) and (a) performed sequentially. For example, the method may also include step (c), indicating that step (c) may be added to the method in any order. For example, the method may include steps (a), (b), and (c), or it may include steps (a), (c), and (b), or it may include steps (c), (a), and (b), etc.

[0035] Unless otherwise specified, the terms "comprising" and "including" as used in this application can be open-ended or closed-ended. For example, "comprising" and "including" can mean that other components not listed may also be included, or that only the listed components may be included.

[0036] Unless otherwise specified, the term "or" is inclusive in this application. For example, the phrase "A or B" means "A, B, or both A and B". More specifically, the condition "A or B" is satisfied by any of the following conditions: A is true (or exists) and B is false (or does not exist); A is false (or does not exist) and B is true (or exists); or both A and B are true (or exist).

[0037] Example 1 This embodiment provides a method for preparing compound A1: ; Specifically, the steps include the following: Step 1: Dissolve compound 1 (5 mmol) in 20 ml of N,N-dimethylformamide, add ammonium chloride (10 mmol) and sodium azide (10 mmol), and stir at 100 °C for 8 h. After the reaction solution cools to room temperature, add dilute hydrochloric acid dropwise to adjust the pH to weakly acidic. A white solid precipitates in the reaction solution. Let it stand at 0 °C overnight, filter and dry the solid to obtain compound 2.

[0038] Step 2: Compound 3 (2 mmol) and compound 4 (2 mmol) were dissolved in 20 ml of tetrahydrofuran, and sodium hydride (2.4 mmol) was slowly added at 0 °C. The mixture was then allowed to react at room temperature for 12 h. The solution was concentrated under reduced pressure and purified by column chromatography to obtain compound 5.

[0039] Step 3: Dissolve compound 5 (2 mmol) in 10 ml of dry dichloromethane, slowly add thionyl chloride (2.4 mmol) dropwise at 0°C, and stir at room temperature for 12 h. Dry the reaction mixture by rotary evaporation, and use the crude product compound 6 directly in the next step.

[0040] Step 4: Compound 6 (0.75 mmol), compound 2 (0.5 mmol), potassium carbonate (1 mmol), and potassium iodide (0.75 mmol) were dissolved in 4 ml of N,N-dimethylformamide and reacted at 60 °C for 3 h. The reaction mixture was extracted three times with ethyl acetate and water, the organic layer was washed with brine, concentrated under reduced pressure, and column chromatography was performed to obtain compound A1.

[0041] Example 2 This embodiment provides a method for preparing compound A2: ; Specifically, the steps include the following: Step 1: Compound 1 (2 mmol) and Compound 2 (2 mmol) were dissolved in 20 ml of tetrahydrofuran, and sodium hydride (2.4 mmol) was slowly added at 0 °C. The mixture was then allowed to react at room temperature for 12 h. The solution was concentrated under reduced pressure and purified by column chromatography to obtain Compound 3.

[0042] Step 2: Dissolve compound 3 (2 mmol) in 10 ml of dry dichloromethane, slowly add thionyl chloride (2.4 mmol) dropwise at 0°C, and stir at room temperature for 12 h. Dry the reaction mixture by rotary evaporation; the crude product, compound 4, can be used directly in the next step.

[0043] Step 3: Compound 5 (0.75 mmol), compound 2 (0.5 mmol), potassium carbonate (1 mmol), and potassium iodide (0.75 mmol) were dissolved in 4 ml of N,N-dimethylformamide and reacted at 60 °C for 3 h. The reaction mixture was extracted three times with ethyl acetate and water, the organic layer was washed with brine, concentrated under reduced pressure, and column chromatography was performed to obtain compound A2.

[0044] Example 3 This embodiment provides a method for preparing compound A3: ; Specifically, the steps include the following: Step 1: Compound 1 (4 mmol) and Compound 2 (4 mmol) were dissolved in 20 ml of tetrahydrofuran, and sodium hydride (4.8 mmol) was slowly added at 0 °C. The mixture was then allowed to react at room temperature for 12 h. The solution was concentrated under reduced pressure and purified by column chromatography to obtain Compound 3.

[0045] Step 2: Dissolve compound 3 (2 mmol) in 10 ml of dry dichloromethane, slowly add thionyl chloride (2.4 mmol) dropwise at 0°C, and stir at room temperature for 12 h. Dry the reaction mixture by rotary evaporation; the crude product, compound 4, can be used directly in the next step.

[0046] Step 3: Compound 5 (0.75 mmol), compound 2 (0.5 mmol), potassium carbonate (1 mmol), and potassium iodide (0.75 mmol) were dissolved in 4 ml of N,N-dimethylformamide and reacted at 60 °C for 3 h. The reaction mixture was extracted three times with ethyl acetate and water, the organic layer was washed with brine, concentrated under reduced pressure, and column chromatography was performed to obtain compound A3.

[0047] Example 4 This embodiment provides a method for preparing compound A4: ; Specifically, the steps include the following: Step 1: Compound 1 (2 mmol) and Compound 2 (2 mmol) were dissolved in 10 ml of tetrahydrofuran solution, and potassium carbonate (6 mmol) was added at 0 °C. The mixture was then stirred at room temperature for 12 h. The solution was concentrated under reduced pressure and purified by column chromatography to obtain Compound 3.

[0048] Step 2: Compound 3 (2 mmol) was dissolved in 10 ml of dry tetrahydrofuran, and lithium aluminum hydride (4 mmol) was slowly added at 0°C. The mixture was then stirred at room temperature for 1 h. 151 mg of water and 151 mg of 10% sodium hydroxide aqueous solution were added at 0°C, followed by 453 mg of water and an appropriate amount of anhydrous magnesium sulfate. The mixture was stirred at room temperature for 15 min. The mixture was filtered, and the filtrate was concentrated under reduced pressure to obtain compound 4.

[0049] Step 3: Dissolve compound 4 (2 mmol) in 10 ml of dry dichloromethane, slowly add thionyl chloride (2.4 mmol) dropwise at 0°C, and stir at room temperature for 12 h. Dry the reaction mixture by rotary evaporation, and use the crude product compound 5 directly in the next step.

[0050] Step 4: Compound 5 (0.75 mmol), compound 6 (0.5 mmol), potassium carbonate (1 mmol), and potassium iodide (0.75 mmol) were dissolved in 4 ml of N,N-dimethylformamide and reacted at 60 °C for 3 h. The reaction mixture was extracted three times with ethyl acetate and water, the organic layer was washed with brine, concentrated under reduced pressure, and column chromatography was performed to obtain compound A4.

[0051] Example 5 This embodiment provides a method for preparing compound A5: ; Specifically, the steps include the following: Step 1: Compound 1 (2 mmol) and Compound 2 (2 mmol) were dissolved in 10 ml of tetrahydrofuran solution, and potassium carbonate (6 mmol) was added at 0 °C. The mixture was then stirred at room temperature for 12 h. The solution was concentrated under reduced pressure and purified by column chromatography to obtain Compound 3.

[0052] Step 2: Compound 3 (2 mmol) was dissolved in 10 ml of dry tetrahydrofuran, and lithium aluminum hydride (4 mmol) was slowly added at 0°C. The mixture was then stirred at room temperature for 1 h. 151 mg of water and 151 mg of 10% sodium hydroxide aqueous solution were added at 0°C, followed by 453 mg of water and an appropriate amount of anhydrous magnesium sulfate. The mixture was stirred at room temperature for 15 min. The mixture was filtered, and the filtrate was concentrated under reduced pressure to obtain compound 4.

[0053] Step 3: Dissolve compound 4 (2 mmol) in 10 ml of dry dichloromethane, slowly add thionyl chloride (2.4 mmol) dropwise at 0°C, and stir at room temperature for 12 h. Dry the reaction mixture by rotary evaporation, and use the crude product compound 5 directly in the next step.

[0054] Step 4: Compound 5 (0.75 mmol), compound 6 (0.5 mmol), potassium carbonate (1 mmol), and potassium iodide (0.75 mmol) were dissolved in 4 ml of N,N-dimethylformamide and reacted at 60 °C for 3 h. The reaction mixture was extracted three times with ethyl acetate and water, the organic layer was washed with brine, concentrated under reduced pressure, and column chromatography was performed to obtain compound A5.

[0055] Example 6 This embodiment provides a method for preparing compound A6: ; Specifically, the steps include the following: Step 1: Compound 1 (2 mmol) and Compound 2 (2 mmol) were dissolved in 40 ml of acetonitrile, and potassium carbonate (6 mmol) was added. The mixture was stirred at 85 °C for 36 h. The solution was concentrated under reduced pressure and purified by column chromatography to obtain Compound 3.

[0056] Step 2: Compound 3 (2 mmol) was dissolved in 10 ml of dry tetrahydrofuran, and lithium aluminum hydride (4 mmol) was slowly added at 0°C. The mixture was then stirred at room temperature for 1 h. 151 mg of water and 151 mg of 10% sodium hydroxide aqueous solution were added at 0°C, followed by 453 mg of water and an appropriate amount of anhydrous magnesium sulfate. The mixture was stirred at room temperature for 15 min. The mixture was filtered, and the filtrate was concentrated under reduced pressure to obtain compound 4.

[0057] Step 3: Dissolve compound 4 (2 mmol) in 10 ml of dry dichloromethane, slowly add thionyl chloride (2.4 mmol) dropwise at 0°C, and stir at room temperature for 12 h. Dry the reaction mixture by rotary evaporation, and use the crude product compound 5 directly in the next step.

[0058] Step 4: Compound 5 (0.75 mmol), compound 6 (0.5 mmol), potassium carbonate (1 mmol), and potassium iodide (0.75 mmol) were dissolved in 4 ml of N,N-dimethylformamide and reacted at 60 °C for 3 h. The reaction mixture was extracted three times with ethyl acetate and water, the organic layer was washed with brine, concentrated under reduced pressure, and column chromatography was performed to obtain compound A6.

[0059] Example 7 This embodiment provides a method for preparing compound A7: ; Specifically, the steps include the following: Step 1: Compound 1 (2 mmol) and Compound 2 (2 mmol) were dissolved in 40 ml of acetonitrile, and potassium carbonate (6 mmol) was added. The mixture was stirred at 85 °C for 36 h. The solution was concentrated under reduced pressure and purified by column chromatography to obtain Compound 3.

[0060] Step 2: Compound 3 (2 mmol) was dissolved in 10 ml of dry tetrahydrofuran, and lithium aluminum hydride (4 mmol) was slowly added at 0°C. The mixture was then stirred at room temperature for 1 h. 151 mg of water and 151 mg of 10% sodium hydroxide aqueous solution were added at 0°C, followed by 453 mg of water and an appropriate amount of anhydrous magnesium sulfate. The mixture was stirred at room temperature for 15 min. The mixture was filtered, and the filtrate was concentrated under reduced pressure to obtain compound 4.

[0061] Step 3: Dissolve compound 4 (2 mmol) in 10 ml of dry dichloromethane, slowly add thionyl chloride (2.4 mmol) dropwise at 0°C, and stir at room temperature for 12 h. Dry the reaction mixture by rotary evaporation, and use the crude product compound 5 directly in the next step.

[0062] Step 4: Compound 5 (0.75 mmol), compound 6 (0.5 mmol), potassium carbonate (1 mmol), and potassium iodide (0.75 mmol) were dissolved in 4 ml of N,N-dimethylformamide and reacted at 60 °C for 3 h. The reaction mixture was extracted three times with ethyl acetate and water, the organic layer was washed with brine, concentrated under reduced pressure, and column chromatography was performed to obtain compound A7.

[0063] Example 8 This embodiment provides a method for preparing compound A8: ; Specifically, the steps include the following: Step 1: Compound 1 (2 mmol), Compound 2 (2 mmol), bis(triphenylphosphine)palladium(II) dichloride (0.01 mmol), N,N-diisopropylethylamine (4 mmol), and cuprous iodide (0.2 mmol) were dissolved in 5 mL of N,N-dimethylformamide and reacted at 100 °C for 6 h under argon protection. After the reaction mixture cooled to room temperature, it was filtered, and the filtrate was extracted three times with ethyl acetate and water. The organic layer was washed with saturated brine. The solution was concentrated under reduced pressure and subjected to column chromatography to obtain Compound 3.

[0064] Step 2: Compound 3 (1 mmol) was dissolved in 10 ml of tetrahydrofuran, and tetrabutylammonium fluoride (2 mmol) was added. The mixture was stirred at room temperature for 2 h. The reaction solution was extracted three times with water and dichloromethane, concentrated under reduced pressure, and subjected to column chromatography to obtain compound 4.

[0065] Step 3: Compound 5 (2 mmol) was dissolved in 10 ml of N,N-dimethylformamide, and sodium azide (2.4 mmol) was added. The mixture was reacted at 90 °C for 10 h. The reaction mixture was extracted with water and ethyl acetate, the organic layer was washed with saturated sodium chloride, concentrated under reduced pressure, and column chromatography was performed to obtain compound 6.

[0066] Step 4: Compound 6 (1 mmol), compound 4 (1 mmol), copper sulfate pentahydrate (0.4 mmol), and sodium ascorbate (10 mmol) were dissolved in a mixture of 4 ml tetrahydrofuran and 1 ml water, and stirred at room temperature for 4 h under argon protection. The mixture was extracted three times with water and dichloromethane, concentrated under reduced pressure, and subjected to column chromatography to obtain compound 7.

[0067] Step 5: Dissolve compounds 7 (0.5 mmol) and 8 (0.6 mmol) in 4 ml of tetrahydrofuran, add potassium tert-butoxide (0.6 mmol), heat to 50 °C and stir for 12 h. Concentrate under reduced pressure and obtain compound A8 by column chromatography.

[0068] Example 9 This embodiment provides a method for preparing compound A9: ; Specifically, the steps include the following: Step 1: Compound 1 (5 mmol) was dissolved in 30 ml of tert-butanol, and di-tert-butyl dicarbonate (7.5 mmol) was added. The mixture was reacted at 90 °C for 12 h. The reaction mixture was extracted with ethyl acetate and water, concentrated under reduced pressure, and subjected to column chromatography to obtain compound 2.

[0069] Step 2: Dissolve A (5 mmol) in 40 ml toluene, add diethyl oxalate (10 mmol), and then add potassium tert-butoxide (10 mmol) in batches at room temperature. Stir for 5 h. The resulting compound 4 does not require further treatment in the reaction solution and can be used as an intermediate in a one-pot reaction to continue to the next step.

[0070] Step 3: Add 80 ml of ethanol and 8 mmol of hydroxylamine hydrochloride to the reaction solution from Step 2 at room temperature and stir for 2 h. Then add 13 ml of water and react for 12 h. Extract repeatedly with ethyl acetate and water, concentrate under reduced pressure to obtain compound 5.

[0071] Step 4: Compound 5 (2 mmol) was dissolved in 10 ml of N,N-dimethylformamide, and triethylamine (4 mmol) was added. The mixture was stirred at 80 °C for 12 h. The mixture was repeatedly extracted with ethyl acetate and water, concentrated under reduced pressure, and then subjected to column chromatography to obtain compound 6.

[0072] Step 5: Dissolve compound 6 (5 mmol) in 10 ml of methanol, add sodium borohydride (10 mmol) at 0 °C, and react at room temperature for 2 h. After the reaction is complete, add an appropriate amount of water to quench the reaction, extract the reaction solution three times with dichloromethane, concentrate under reduced pressure, and obtain compound 7 by column chromatography.

[0073] Step 6: Compound 7 (5 mmol) was dissolved in 4 ml of N,N-dimethylacetamide, and benzotriazole (10 mmol) and thionyl chloride (10 mmol) were added at 0 °C. The reaction mixture was stirred for 12 h, and extracted three times with ethyl acetate and water. The ethyl acetate layer was removed. The pH was adjusted to neutral with sodium bicarbonate aqueous solution, and the mixture was extracted three times with ethyl acetate. The solution was concentrated under reduced pressure to obtain compound 8.

[0074] Step 7: Compound 8 (2 mmol) was dissolved in 5 ml of N,N-dimethylformamide, and sodium azide (3 mmol) was added. The mixture was stirred at 90 °C for 12 h. The reaction mixture was extracted with ethyl acetate and water, the organic layer was washed with saturated sodium chloride, concentrated under reduced pressure, and column chromatography was performed to obtain compound 9.

[0075] Step 8: Compound 9 (1 mmol), compound 10 (1 mmol), copper sulfate pentahydrate (0.4 mmol), and sodium ascorbate (10 mmol) were dissolved in a mixture of 4 ml tetrahydrofuran and 1 ml water, and stirred at room temperature for 4 h under argon protection. The mixture was extracted three times with water and dichloromethane, concentrated under reduced pressure, and subjected to column chromatography to obtain compound 11.

[0076] Step 9: Dissolve compounds 11 (0.5 mmol) and 12 (0.6 mmol) in 4 ml of tetrahydrofuran, add potassium tert-butoxide (0.6 mmol), heat to 50 °C and stir for 12 h. Concentrate under reduced pressure and obtain compound A9 by column chromatography.

[0077] Example 10 This embodiment provides a method for preparing compound A10: ; Specifically, the steps include the following: Step 1: Compound 1 (10 mmol) was dissolved in 30 ml of dichloromethane, and di-tert-butyl dicarbonate (15 mmol) and 4-dimethylaminopyridine (1 mmol) were added. The mixture was stirred at room temperature for 12 h. After the reaction was completed, the solution was concentrated under reduced pressure and column chromatography was performed to obtain compound 2.

[0078] Step 2: Compound 2 (5 mmol) and compound 3 (5 mmol) were dissolved in 20 ml of acetonitrile, and potassium carbonate (10 mmol) was added. The mixture was stirred at room temperature for 12 h. The solution was concentrated under reduced pressure and then subjected to column chromatography to obtain compound 4.

[0079] Step 3: Compound 4 (1 mmol) was dissolved in anhydrous formic acid (4 ml), and the reaction was stirred at room temperature for 12 h. The solution was concentrated under reduced pressure and purified by column chromatography to obtain compound 6.

[0080] Step 4: Compound 6 (0.2 mmol) and compound 7 (0.4 mmol) were dissolved in 2 ml of tetrahydrofuran, and potassium tert-butoxide (0.4 mmol) was added. The mixture was reacted at 50 °C for 12 h. The solution was concentrated under reduced pressure and purified by column chromatography to obtain compound A10.

[0081] Example 11 This embodiment provides a method for preparing compound A11: ; Specifically, the steps include the following: Step 1: Compound 1 (10 mmol) was dissolved in dry methanol (80 mmol) solution. The reaction flask was sealed with a rubber stopper, and acetyl chloride (120 mmol) was slowly added dropwise at 0°C. The mixture was stirred at 0°C for 24 h. After the reaction was complete, the gas was slowly released, the pH was adjusted to neutral with sodium bicarbonate, and the reaction solution was extracted three times with water and dichloromethane. The solution was concentrated under reduced pressure and obtained by column chromatography to yield compound 2.

[0082] Step 2: Dissolve compound 2 (5 mmol) and compound 3 (6 mmol) in 10 ml of ethanol and stir at room temperature for 12 h. Concentrate under reduced pressure, and use the crude product compound 4 directly in the next step.

[0083] Step 3: Compound 5 (5 mmol) and compound 6 (5 mmol) were dissolved in 10 ml of tetrahydrofuran, and potassium tert-butoxide (5 mmol) was slowly added at 0 °C. The mixture was then stirred at room temperature for 6 h. The solution was concentrated under reduced pressure and purified by column chromatography to obtain compound 7.

[0084] Step 4: Compound 7 (0.5 mmol), compound 4 (0.75 mmol), cuprous iodide (0.1 mmol), potassium carbonate (2 mmol), and tetrakis(triphenylphosphine)palladium (0.05 mmol) were dissolved in 5 ml of N,N-dimethylformamide and stirred at 60 °C for 4 h under argon protection. The reaction mixture was extracted multiple times with ethyl acetate and water, the organic layer was washed with saturated brine, concentrated under reduced pressure, and column chromatography was used to obtain compound A11.

[0085] Example 12 This embodiment provides a method for preparing compound A12: ; Specifically, the steps include the following: Step 1: Compound 1 (10 mmol) and Compound 2 (10 mmol) were dissolved in 30 ml of tetrahydrofuran, and potassium carbonate (15 mmol) was added at 0 °C. The mixture was then stirred at room temperature for 12 h. The solution was concentrated under reduced pressure and purified by column chromatography to obtain Compound 3.

[0086] Step 2: Compound 3 (6 mmol), compound 4 (5 mmol), 1,1'-bis(diphenylphosphino)ferrocene palladium(II) dichloride (0.5 mmol), and potassium carbonate (15 mmol) were dissolved in a mixed solution of 10 ml tetradioxane and 1 ml water. The reaction was carried out at 100 °C for 5 h under argon protection. The reaction solution was filtered, the filtrate was concentrated under reduced pressure, and compound 5 was obtained by column chromatography.

[0087] Step 3: Compound 5 (0.5 mmol) was dissolved in anhydrous formic acid (2 ml), and the reaction was stirred at room temperature for 12 h. The solution was concentrated under reduced pressure and column chromatography was used to obtain compound A12.

[0088] Biological testing I. Antibacterial Concentration Test 1. Experimental Objective The minimum inhibitory concentrations (MICs) of the compounds prepared in Examples 1-11 against fungi were tested.

[0089] 2. Experimental strains and test culture media Experimental strain: Candida albicans BNCC186382; Test medium: YM medium (Yeast Malt Agar).

[0090] 3. Experimental Procedure 3.1. Preparation of compound masterbatch The compound was first prepared into a 3.2 mg / mL stock solution using DMSO and stored at -20°C until use.

[0091] Add 10 μL of the stock solution to well 2 of a 96-well microplate (flat bottom). Dilute it 2-fold with DMSO to prepare 0.19 μg / mL of 100x working solution (Upper region: Wells 2-11: 1600, 800, 400, 200, 100, 50, 25, 12.5, 6.25, 3.125; Lower region: Wells 2-5: 1.56, 0.78, 0.39, 0.19) (The 96-well plate is divided into two regions: ABCD is the upper region, and EFGH is the lower region). 100% DMSO is used as a positive control. This is the compound master plate.

[0092] 3.2. Preparation of inoculum Streaking the -80℃ frozen bacteria Candida albicans ATCC MYA-2876 onto SDA plates and incubating aerobically at 28℃ for 48 hours. Remove the plates, pick 2-3 single colonies from the plate and transfer them to 50 mL of sterile water, then dilute 10-fold with YM medium. This bacterial suspension contains 2.5 × 10³ CFU / mL. This is the inoculum.

[0093] 3.3. MIC Detection Transfer 2 μL of 100x working solution from the compound master plate (3.1 preparation) to a flat-bottomed 96-well plate (containing 198 μL of inoculum (3.2 preparation)) to obtain the MIC test plate. Therefore, the final test concentrations of the compounds were 16, 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.0625, 0.03125, 0.0156, 0.0078, 0.0039, and 0.0019 μg / mL. 1% DMSO was used as a growth control. All test plates were incubated aerobically at 28°C for 24–48 h.

[0094] 3.4. Reading the MIC After culturing, determine the MIC (μg / mL) of the test compound against yeast by visual observation according to the standards in the table below.

[0095] The MIC criteria for the test compounds against fungi are shown in Table 1.

[0096] Table 1 .

[0097] 4. Experimental Results Table 2

[0098] .

[0099] Note: The experimental results are from three independent experiments, and the unit is μg / mL.

[0100] A: MIC≤0.05; B: 0.010<MIC≤ 1.0; C: 1.0≤MIC<10.0.

[0101] As can be seen from Table 1, the compounds provided by this invention have good inhibitory activity against Candida.

[0102] Liver microsomal stability test 1. Experimental Procedure 1.1 Materials NADPH system A / B solutions were rapidly dissolved in a water bath at 37°C and then kept on ice for later use; liver microsomes were dissolved on ice for later use.

[0103] 1.2 Preparation of inactivation incubation solution Take an appropriate amount of rat liver microsomal solution, evaporate to dryness at 100℃, replenish with PBS, sonicate to disrupt, and prepare various genera inactivation incubation systems (prepare 1 ml of 940 μl + 50 μl + 10 μl + 5 μl) according to the volume ratio of PBS buffer, Solution A, Solution B and liver microsomal dilution solution of 94:5:1:0.5.

[0104] Preparation of standard curves Standard stock solutions: 0.1, 0.5, 1, 2, 5, 10 mM compound stock solutions. Take 10 μL of the solution and add 90 μL of the inactivation system to obtain the final concentrations of 10, 50, 100, 200, 1000 μM working solution. Add 250 μL of ice-cold methanol to terminate the reaction. Centrifuge at 13000 rpm for 5 min at 4 °C. Take 20 μL of the supernatant for injection.

[0105] 1.4 Compound Preparation Compounds: Take 50 μL of Solution A, 10 μL of Solution B, and 940 μL of PBS buffer. Take 990 μL and add 10 μL of the corresponding compound (final concentration 200 μM). Vortex to mix, and briefly centrifuge at 4°C to allow the liquid on the tube wall to reach the bottom. Incubate at 37°C for 5 min, then add 5 μL of liver microsomes. After incubation for 0, 15, 30, 45, 60, 90, 120, 180, and 270 min, respectively, remove 100 μL and immerse it in 250 μL of ice-cold methanol to stop the reaction. Immediately vortex for 3 min, centrifuge at 13000 rpm for 10 min at 4°C, and inject 20 μL of the supernatant to determine the content of the analyte. A blank control containing drug-inactivated liver microsomes is also set up.

[0106] The above experiments show that the compound has better stability in liver microsomes, and it is expected to be administered to humans at a lower frequency and with a lower dose.

[0107] Finally, it should be emphasized that the specific embodiments described in this invention can be improved and modified in various ways by those skilled in the art without departing from the principles of this invention, according to actual needs. Therefore, the scope of protection of this invention is not limited to the specific content of the above embodiments, but should be determined by the scope defined in the claims. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this invention should be included within the scope of protection of this invention.

Claims

1. A five-membered heteroaryl antifungal compound, characterized in that, The compound is a compound with the structural formula shown in formula (1) or a pharmaceutically acceptable salt, prodrug, or isotopic derivative thereof: ； （1） Among them, R1 is selected from F and H; R2 is selected from F and H; R3 is selected from NO2, CN, and H; R4 is selected from CN and H; R5 is selected from CN, F, and H; L1 is selected from -CH2O-, -OCH2- and -O-; T1 is selected from N and CH.

2. The five-membered heteroaryl antifungal compound according to claim 1, characterized in that, The structural formula of the compound is shown in A1 below: ； Where Ar is selected from R1 is selected from H; R2 is selected from H; R3 is selected from H; R4 is selected from CN; R5 is selected from H; L1 is selected from -CH2O-; T1 is selected from N.

3. The five-membered heteroaryl antifungal compound according to claim 1, characterized in that, The structural formula of the compound is shown in A2 below: ； Where Ar is selected from R1 is selected from H; R2 is selected from H; R3 is selected from H; R4 is selected from H; R5 is selected from CN; L1 is selected from -CH2O-; T1 is selected from N.

4. The five-membered heteroaryl antifungal compound according to claim 1, characterized in that, The structural formula of the compound is shown in A3 below: ； Where Ar is selected from R1 is selected from H; R2 is selected from H; R3 is selected from NO2; R4 is selected from H; R5 is selected from H; L1 is selected from -CH2O-; T1 is selected from N.

5. The five-membered heteroaryl antifungal compound according to claim 1, characterized in that, The structural formula of the compound is shown in A4 below: ； Where Ar is selected from R1 is selected from H; R2 is selected from F; R3 is selected from H; R4 is selected from H; R5 is selected from H; L1 is selected from -OCH2-; T1 is selected from CH.

6. The five-membered heteroaryl antifungal compound according to claim 1, characterized in that, The structural formula of the compound is shown in A5 below: ； Where Ar is selected from R1 is selected from F; R2 is selected from H; R3 is selected from H; R4 is selected from H; R5 is selected from H; L1 is selected from -OCH2-; T1 is selected from CH.

7. The five-membered heteroaryl antifungal compound according to claim 1, characterized in that, The structural formula of the compound is shown in A6 below: ； Where Ar is selected from R1 is selected from F; R2 is selected from H; R3 is selected from H; R4 is selected from H; R5 is selected from F; L1 is selected from -O-; T1 is selected from N.

8. The five-membered heteroaryl antifungal compound according to claim 1, characterized in that, The structural formula of the compound is shown in A7 below: ； Where Ar is selected from R1 is selected from H; R2 is selected from F; R3 is selected from H; R4 is selected from H; R5 is selected from F; L1 is selected from -O-; T1 is selected from N.

9. The five-membered heteroaryl antifungal compound according to claim 1, characterized in that, The structural formula of the compound is shown in A8 below: ； Where Ar is selected from R1 is selected from H; R2 is selected from H; R3 is selected from H; R4 is selected from H; R5 is selected from H; L1 is selected from -CH2O-; T1 is selected from N.

10. The five-membered heteroaryl antifungal compound according to claim 1, characterized in that, The structural formula of the compound is shown in A11 below: ； Where Ar is selected from R1 is selected from H; R2 is selected from H; R3 is selected from CN; R4 is selected from H; R5 is selected from H; L1 is selected from -CH2O-; T1 is selected from N.

11. The application of the five-membered heteroaryl antifungal compound according to any one of claims 1 to 10, characterized in that, The five-membered heteroaryl compounds are used to inhibit the activity of fungal Gwt1 protein.

12. The application of the five-membered heteroaryl antifungal compound according to claim 11, characterized in that, The fungi include Candida, Aspergillus, and Cryptococcus.

13. The application of the five-membered heteroaryl antifungal compound according to claim 11, characterized in that, Used to prepare drugs for treating deep fungal infections.

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